Peptide ketoamides

ABSTRACT

A novel class of peptide α-ketoamides useful for selectively inhibiting serine proteases, selectively inhibiting cysteine proteases, generally inhibiting all serine proteases, and generally inhibiting all cysteine proteases, having the formula M 1  --AA--NH--CHR 2  --CO--CO--NR 3  R 4 , M 1  --AA 2  --AA 1  --CO--NR 3  R 4 , M 1  --AA--AA--AA--CO--NR 3  R 4 , M 1  --AA--AA--AA--AA--CO--NR 3  R 4 , or M 1  --AA--CO--NR 3  R 4 .

This is a continuation of application Ser. No. 08/246,511 filed on May20, 1994, now abandoned, which is a continuation of Ser. No. 08/118,997filed on Sep. 9, 1993, now abandoned, which is a continuation of Ser.No. 07/815,073 filed on Dec. 27, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel class of peptide ketoamides useful forselectively inhibiting serine proteases, selectively inhibiting cysteineproteases, generally inhibiting all serine proteases, and generallyinhibiting all cysteine proteases. Serine proteases and cysteineproteases are involved in numerous disease states and inhibitors forthese enzymes can be used therapeutically for the treatment of diseasesinvolving serine proteases or cysteine proteases. We have discoveredthat peptide α-ketoamides can be constructed to inhibit selectivelyindividual serine or cysteine proteases or groups of serine or cysteineproteases. We have found that peptide ketoamides which containhydrophobic aromatic amino acid residues in the P₁ site are potentinhibitors of chymases and chymotrpysin-like enzymes. Ketoamidescontaining small hydrophobic amino acid residues at the P₁ position aregood inhibitors of elastases. Inhibitors of elastases and chymases areuseful as anti-inflammatory agents. We show that peptide ketoamideswhich contain cationic amino acid residues such as Arg and Lys in the P₁site will be potent inhibitors of trypsin and blood coagulation enzymes.These inhibitors are thus useful as anticoagulants. Cysteine proteasessuch as papain, cathepsin B, and calpain I and II are also inhibited byketoamides. Ketoamides with aromatic amino acid residues in the P₁ siteare good inhibitors for cathepsin B and papain. Thus, they would haveutility as anticancer agents. Ketoamides with either aromatic amino acidresidues or small hydrophobic alkyl amino acid residues at P₁ are goodinhibitors of calpain I and II. These inhibitors are useful asneuroprotectants and can be used as therapeutics for the treatment ofneurodegeneration and stroke.

2. Nomenclature

In discussing the interactions of peptides with serine and cysteineproteases, we have utilized the nomenclature of Schechter and Berger[Biochem. Biophys. Res. Commun. 27, 57-162 (1967); incorporated hereinby reference]. The individual amino acid residues of a substrate orinhibitor are designated P₁, P₂, etc. and the corresponding subsites ofthe enzyme are designated S₁, S₂, etc. The scissile bond of thesubstrate is S₁ --S₁ '. The primary substrate recognition site of serineproteases is S₁. The most important recognition subsites of cysteineproteases are S₁ and S₂.

Amino acid residues and blocking groups are designated using standardabbreviations [see J. Biol. Chem. 260, 14-42 (1985) for nomenclaturerules; incorporated herein by reference]. An amino acid residue (AA) ina peptide or inhibitor structure refers to the part structure --NH--CHR₁--CO--, where R₁ is the side chain of the amino acid residue AA. Apeptide α-ketoester residue would be designated --AA--CO--OR whichrepresents the part structure --NH--CHR₁ --CO--CO--OR. Thus, the ethylketoester derived from benzoyl alanine would be designatedBz--Ala--CO--OEt which represents C₆ H₅ CO--NH--CHMe--CO--CO--OEt.Peptide ketoamide residues would be designated --AA--CO--NH--R. Thus,the ethyl ketoamide derived from Z--Leu--Phe--OH would be designatedZ--Leu--Phe--CO--NH--Et which represents C₆ H₅ CH₂ OCO--NH--CH(CH₂CHMe₂)--CO--NH--CH(CH₂ Ph)--CO--CO--NH--Et.

3. Description of the Related Art

Cysteine Proteases. Cysteine proteases such as calpain use a cysteineresidue in their catalytic mechanism in contrast to serine proteaseswhich utilize a serine residue. Cysteine proteases include papain,cathepsin B, calpains, and several vital enzymes. Neural tissues,including brain, are known to possess a large variety of proteases,including at least two calcium stimulated proteases termed calpains.Calpains are present in many tissues in addition to the brain. Calpain Iis activated by micromolar concentrations of calcium while calpain II isactivated by millimolar concentrations. In the brain, calpain II is thepredominant form, but calpain I is found at synaptic endings and isthought to be the form involved in long term potentiation, synapticplasticity, and cell death. Other Ca²÷ activated cysteine proteases mayexist, and the term "calpain" is used to refer to all Ca²⁺ activatedcysteine proteases, including calpain I and calpain II. The terms"calpain I" and "calpain II" are used herein to refer to the micromolarand millimolar activated calpains, respectively, as described above.While calpains decade a wide variety of protein substrates, cytoskeletalproteins seem to be particularly susceptible to attack In some cases,the products of the proteolytic digestion of these proteins by calpainare distinctive and persistent over time. Since cytoskeletal proteinsare major components of certain types of cells, this provides a simplemethod of detecting calpain activity in cells and tissues. Thus, calpainactivation can be measured indirectly by assaying the proteolysis of thecytoskeletal protein spectrin, which produces a large, distinctive andbiologically persistent breakdown product when attacked by calpain[Siman, Bandry, and Lynch, Proc. Natl. Acad. Sci. USA 81, 3572-3576(1984); incorporated herein by reference]. Activation of calpains and/oraccumulation of breakdown products of cytoskeletal elements has beenobserved in neural tissues of mammals exposed to a wide variety ofneurodegenerative diseases and conditions. For example, these phenomenahave been observed following ischemia in gerbils and rats, followingstroke in humans, following administration of the toxins kainate,trimethyltin or colchicine in rats, and in human Alzheimer's disease.

Several inhibitors of calpain have been described including peptidealdehydes such as Ac--Leu--Leu--Nle--H and leupeptin(Ac--Leu--Leu--Arg--H), as well as epoxysuccinates such as E-64. Thesecompounds are not especially useful at inhibiting calpain in neuraltissue in vivo because they are poorly membrane permeant and,accordingly, are not likely to cross the blood brain barrier very well.Also, many of these inhibitors have poor specificity and will inhibit awide variety of proteases in addition to calpain. Other classes ofcompounds which inhibit cysteine proteases include peptide diazomethylketone (Rich, D. H., in Protease Inhibitors, Barrett A. I., andSalversen, G., Eds., Elsevier, New York, 1986, pp 153-178; incorporatedherein by reference). Peptide diazomethyl ketones are potentiallycarcinogenic and are thought to be poorly membrane permeant and to havelow specificity. Thus, no effective therapy has yet been developed formost neurodegenerative diseases and conditions. Millions of individualssuffer from neurodegenerative diseases and thus, there is a need fortherapies effective in treating and preventing these diseases andconditions.

Cathepsin B is involved in muscular dystrophy, myocardial tissue damage,tumor metastasis, and bone resorption. In addition, a number of vitalprocessing enzymes, which are essential for vital infection, arecysteine proteases. Inhibitors of cysteine proteases would have multipletherapeutic uses.

Serine Proteases. Serine proteases play critical roles in severalphysiological processes such as digestion, blood coagulation, complementactivation, fibrinolysis, vital infection, fertilization, andreproduction. Serine proteases are not only a physiological necessity,but also a potential hazard if they are not controlled. Uncontrolledproteolysis by elastases may cause, pancreatitis, emphysema, rheumatoidarthritis, bronchial inflammation and adult respiratory distresssyndrome. It has been suggested that a new trypsin-like cellular enzyme(tryptase) is involved in the infection of human immunodeficiency virustype 1 [HIV-1; Hattori et al., FEBS Letters 248, pp. 48-52 (1989)],which is a causative agent of acquired immunodeficiency syndrome (AIDS).Plasmin is involved in tumor invasiveness, tissue remodeling,blistering, and clot dissociation. Accordingly, specific and selectiveinhibitors of these proteases should be potent anticoagulants,anti-inflammatory agents, anti-tumor agents and anti-viral agents usefulin the treatment of protease-related diseases [Powers and Harper,Proteinase Inhibitors, pp 55-152, Barrett and Salvesen, eds., Elsevier,(1986); incorporated herein by reference]. In vitro proteolysis bychymotrypsin, trypsin or the elastase family is a serious problem in theproduction, purification, isolation, transport or storage of peptidesand proteins.

Elastase inhibitors are anti-inflammatory agents which can be used totreat elastase-associated inflammation including rheumatoid arthritisand emphysema. Although the naturally occurring protease inhibitor,α1-protease inhibitor (α1-PI) has been used to treat patients withemphysema, this protein inhibitor is not widely used clinically due tothe high dosage needed for treatment and the difficulty of producinglarge quantities. Therefore, small molecular weight elastase inhibitorsare needed for therapy. Other low molecular weight elastase inhibitorshave utility for the treatment of emphysema and intimation (see:1-carpapenem-3-carboxylic esters as anti-inflammatory agents, U.S. Pat.No. 4,493,839; N-carboxyl-thienamycin esters and analogs thereof asanti-inflammatory agents, U.S. Pat. No. 4,495,197; incorporated hereinby reference).

Anticoagulants and antithrombotic drugs are used in a variety ofthrombotic disorders. The 1990 Physician's Desk Reference lists severalanticoagulant drugs (heparin, protamine sulfate and warfarin), a fewantiplatelet drugs (aspirin) and several thrombolytic agents. Herringand warfarin are commonly used clinically for prevention and treatmentof venous thrombosis and pulmonary embolism. Heparin inhibits the bloodcoagulation activity by accelerating the binding of natural plasmaprotease inhibitor antithrombin Ill with coagulation factors, andwarfarin acts as a vitamin K antagonist and inhibits the synthesis ofcoagulation factors. None of the anticoagulant drugs, antithromboticdrugs, fibrolytic agents and antiplatelet drags are highly effective inall clinical situations and many induce side reactions [Von Kaulla,Burger's Medicinal Chemistry, Part II, pp 1081-1132, Wolff, ed., (1979);incorporated herein by reference]. Coagulation disorders such asdisseminated intravascular coagulation, bleeding complications ofmedical and surgical procedures and bleeding complications of systemicillness are still difficult to manage [Ingram, Brozovic and Slater,Bleeding Disorders, pp 1-413, Blackwell Scientific Publications, (1982);incorporated herein by reference]. In the treatment of patients withcoagulation problems, anticoagulant or antithrombotic agents of diversemechanisms are urgently sought in order to provide better medical care.Inhibitors for the trypsin-like enzymes involved in blood coagulationare useful anticoagulants in vivo [see for example:H--D--Phe--Pro--Arg--CH₂ Cl, Hanson and Harker, Proc. Natl. Acad. Sci.85, 3184-3188 (1988);7-Amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin (ACITIC), Oweida,Ku, Lumsden, Karn, and Powers, Thrombos. Res. 58, 191-197 (1990);incorporated herein by reference].

Ketoesters. A few amino acid and peptide ketoesters and ketoacids havebeen previously reported. Cornforth and Cornforth [J. Chem. Soc., 93-96(1953); incorporated herein by reference] report the synthesis of theketoacids PhCH₂ CO--Gly--CO--OH and Ac--Gly--CO--OH upon hydrolysis ofheterocyclic molecules. Charles et al. [J. Chem. Soc. Perkin I, 139-1146(1980); incorporated herein by reference] use ketoesters for thesynthesis of bicyclic heterocycles. They report the synthesis ofn--BuCO--Ala--CO--OEt, PrCO--Ala--CO--OEt, cyclopentylCO--Ala--CO--OEt,PrCO--PhGly--CO--OEt, and Bz--Ala--CO--OEt. Hori et al. [Peptides:Structure and Function-Proceedings of the Ninth American PeptideSymposium (Deber, Hruby, and Kopple, Eds.) Pierce Chemical Co., pp819-822 (1985); incorporated herein by reference] reportBz--Ala--CO--OEt, Bz--Ala--CO--OH, Z--Ala--Ala--Abu--CO--OEt,Z--Ala--Ala--Abu--CO--OBz], and Z--Ala--Ala--Ala--Ala--CO--OEt(Abu=2-aminobutanoic acid or α-aminobutyric acid) and report that thesecompounds inhibit elastase. Trainer [Trends Pharm. Sci. 8, 303-307(1987); incorporated herein by reference] comments on one of thiscompounds. Burkhart, J., Peet, N. P., and Bey, P. [Tetrahedron Left. 29,3433-3436 (1988); incorporated herein by reference] report the synthesisof Z--Val--Phe--CO--OMe and Bz--Phe--CO--OMe.

Mehdi et al., [Biochem. Biophys. Res. Comm. 166, 595-600 (1990);incorporated herein by reference] report the inhibition of humanneutrophil elastase and cathepsin G by peptide α-ketoesters. Angelastroet al., [J. Med. Chem. 33, 13-16 (1990); incorporated herein byreference] report some α-ketoesters which are inhibitors of calpain andchymotrypsin. Hu and Abeles [Arch. Biochem. Biophys. 281, 271-274(1990)]; incorporated herein by reference] report some peptidylα-ketoamides and α-ketoacids which are inhibitors of cathepsin B andpapain. Peet et al. [J. Med. Chem. 33, 394-407 (1990); incorporatedherein by reference] report some peptidyl α-ketoesters which areinhibitors of porcine pancreatic elastase, human neutrophil elastase,and rat & human neutrophil cathepsin G.

Ketoamides. A single peptide ketoamide is reported in the literature byHu and Abeles [Arch. Biochem. Biophys. 281, 271-274 (1990)]. Thiscompound Z--Phe--NHCH₂ CO--CO--NH--Et or Z--Phe--Gly--CO--NH--Et isreported to be an inhibitor of papain (K_(I) =1.5 μM) and cathepsin B(K_(I) =4 μM).

SUMMARY OF THE INVENTION

We have discovered that peptide and amino acid α-ketoamide derivativesare a novel group of inhibitors for serine proteases and cysteineproteases. Inhibitors are compounds that reduce or eliminate thecatalytic activity of the enzyme. We have discovered that peptideketoamide derivatives, which have an amino acid sequence similar to thatof good substrates for a particular protease, are good inhibitors forthat protease. Thus, we are able to predict the structures of newinhibitors for other serine and cysteine proteases based on knowledge oftheir substrate specificities.

We have discovered some peptide α-ketoamide derivatives which arespecific inhibitors for chymotrypsin. Chymotrypsin and chymotrypsin-likeenzymes hydrolyze peptide bonds where P₁ amino acid is Trp, Tyr, Phe,Met, Leu or other amino acid residues which contain aromatic or largealkyl side chains. Inhibitors with these residues at P₁ are goodchymotrypsin and chymase inhibitors. Trypsin and trypsin-like enzymesnormally cleave peptide bonds in proteins and peptides where the aminoacid residue on the carbonyl side of the split bond (P₁ residue) is Lysor Arg. We show that peptide α-ketoamide derivatives which have Lys orArg at P₁ will be good inhibitors for these enzymes. Elastase andelastase-like enzymes cleave peptide bonds where the P₁ amino acid isAla, Val, Set, Leu and other similar amino acids. We shown thatinhibitors with these residues at P₁ are good elastase inhibitors. Allof the above enzymes have extensive secondary specificity and recognizeamino acid residues removed from the P₁ residue.

The peptide α-ketoamide derivatives are also novel and potent inhibitorsof cysteine proteases including calpains and cathepsin B. The calpaininhibitors are useful for treatment of various neurodegenerativediseases and conditions, including ischemia, stroke, and Alzheimer'sdisease.

The new protease inhibitors, especially the elastase inhibitors, trypsininhibitors, and chymase inhibitors are useful for controlling tissuedamage and various inflammatory conditions mediated by proteases such asblistering. The inhibitors for blood coagulation enzymes will be usefulanticoagulants and could be used to treat thrombosis.

The peptide and amino acid α-ketoamide derivatives are also useful invitro for inhibiting trypsin, elastase, chymotrypsin and other serineproteases of similar specificity, and for inhibiting serine proteases ingeneral. The inhibitors can be used to identify new proteolytic enzymesencountered in research. They can also be used in research andindustrially to prevent undesired proteolysis that occurs during theproduction, isolation, purification, transport and storage of valuablepeptides and proteins. Such proteolysis often destroys or alters theactivity and/or function of the peptides and proteins. Uses wouldinclude the addition of the inhibitors to antibodies, enzymes, plasmaproteins, tissue extracts or other proteins and peptides which arewidely sold for use in clinical analyses, biomedical research, and formany other reasons. For some uses a specific inhibitor would bedesirable, while in other cases, an inhibitor with general specificitywould be preferred.

DETAILED DESCRIPTION OF THE INVENTION

Peptide α-ketoamides are transition state analog inhibitors for serineproteases and cysteine proteases. Peptide α-ketoamides containinghydrophobic amino acid residues in the P₁ site have been found to beexcellent inhibitors of serine proteases including porcine pancreaticelastase and bovine chymotrypsin. We show that peptide α-ketoamidescontaining amino acid residues with cationic side chains in the P₁ sitewill be excellent inhibitors of several serine proteases includingbovine trypsin, bovine thrombin, human plasma kallikrein, porcinepancreatic kallikrein, human factor XIa and human plasmin. Peptideα-ketoamides containing amino acid residues with hydrophobic side chainat the P₁ site have also been found to be excellent inhibitors ofseveral cysteine proteases including papain, cathepsin B, calpain I, andcalpain II. These structures may be used in vivo to treat diseases suchas emphysema, adult respiratory distress syndrome, rheumatoid arthritisand pancreatitis which result from uncontrolled proteolysis by elastase,chymotrypsin, trypsin and related serine proteases. These inhibitors maybe used in vitro to prevent proteolysis which occurs in the process ofproduction, isolation, purification, storage or transport of peptidesand proteins. These inhibitors may be useful as therapeutic agents fortreatment of neurodegeneration, viral infections, muscular dystrophy,myocardial tissue damage, tumor metastasis, and bone resorption.

The novel class of dipeptide α-ketoamides have the following structuralformula:

    M.sub.1 --AA--NH--CHR.sub.2 --CO--CO--NR.sub.3 R.sub.4

or a pharmaceutically acceptable salt, wherein

M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂ --, X--NH--CO--, X₂N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂ N--SO₂ --, X--CO--,X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--;

X is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀fluoroalkyl, C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkylsubstituted with I, 1-admantyl, 9-fluorenyl, phenyl, phenyl substitutedwith K, phenyl disubstituted with K, phenyl trisubstituted with K,naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,naphthyl trisubstituted with K, C₁₋₁₀ alkyl with an attached phenylgroup, C₁₋₁₀ alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with anattached phenyl group substituted with K, C₁₋₁₀ alkyl with two attachedphenyl groups substituted with K, C₁₋₁₀ alkyl with an attached phenoxygroup, and C₁₋₁₀ alkyl with an attached phenoxy group substituted with Kon the phenoxy group;

J is selected from the group consisting of halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine, C₁₋₁₀alkyl-0--CO--, C₁₋₁₀ alkyl--O--CO--NH--, and C₁₋₁₀ alkyl-S--;

K is selected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfluoroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁ -C₁₀ acyl, and C₁₋₁₀ alkoxy-CO--, andC₁₋₁₀ alkyl-S--;

AA is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic add, glutamic acid, lysine, arginine, histidine, phenylglycine,beta-alanine, norleucine, norvaline, alpha-aminobutyric acid,epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ -CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)--COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine;

R₂ is selected from the group consisting of C₁₋₈ branched and unbranchedalkyl, C₁₋₈ branched and unbranched cyclized alkyl, and C₁₋₈ branchedand unbranched fluoroalkyl;

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₃₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ring attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, C₁₋₁₀ with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

The novel class of dipeptide α-ketoamides also have the followingstructural formula:

    M.sub.1 --AA.sub.2 --AA.sub.1 --CO--NR.sub.3 R.sub.4

or a pharmaceutically acceptable salt, wherein

M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂ --, X--NH--CO--, X₂N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂ N--SO₂ --, X--CO--,X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--;

X is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀fluoroalkyl, C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkylsubstituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substitutedwith K, phenyl disubstituted with K, phenyl trisubstituted with K,naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,naphthyl trisubstituted with K, C₁₋₁₀ alkyl with an attached phenylgroup, C₁₋₁₀ alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with anattached phenyl group substituted with K, C₁₋₁₀ alkyl with two attachedphenyl groups substituted with K, C₁₋₁₀ alkyl with an attached phenoxygroup, and C₁₋₁₀ alkyl with an attached phenoxy group substituted with Kon the phenoxy group;

J is selected from the group consisting of halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine, C₁₋₁₀alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--;

K is selected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfloroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀ alkylamino, C₂₋₁₂ dialkylamino, C₁ -C₁₀ acyl, and C₁₋₁₀ alkoxy-CO--, andC₁₋₁₀ alkyl-S--;

AA₁ is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparticacid, glutamic acid, lysine, arginine, histidine, phenylglycine,beta-alanine, norleucine, norvaline, alpha-aminobutyric acid,epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ -CH(CH₂ CHEt2)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂-cyclopropyl)-COOH, trifluoroleucine, and hexafluoroleucine;

AA₂ is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH)CH₂ -cyclohexyl) -COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine;

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₃₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ring attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, C₁₋₁₀ with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

The novel class of tripeptide α-ketoamides have the following structuralformula:

    M.sub.1 --AA--AA--AA--CO--NR.sub.3 R.sub.4

or a pharmaceutically acceptable salt, wherein

M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂ --, X--NH--CO--, X₂N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X--SO₂ --, X--CO--,X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--;

X is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀fluoroalkyl, C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkylsubstituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substitutedwith K, phenyl disubstituted with K, phenyl trisubstituted with K,naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,naphthyl trisubstituted with K, C₁₋₁₀ alkyl with an attached phenylgroup, C₁₋₁₀ alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with anattached phenyl group substituted with K, C₁₋₁₀ alkyl with two attachedphenyl groups substituted with K, C₁₋₁₀ alkyl with an attached phenoxygroup, and C₁₋₁₀ alkyl with an attached phenoxy group substituted with Kon the phenoxy group;

J is selected from the group consisting of halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine, C₁₋₁₀alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--;

K is selected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfluoroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁₋₁₀ acyl, and C₁₋₁₀ alkoxy-CO--, andC₁₋₁₀ alkyl-S--;

AA is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinencarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂₋₂ -napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂ -CH(CH₂-cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH, trifluoroleucine,and hexafluoroleucine;

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₃₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ting attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, C₁₋₁₀ with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

The novel class of tetrapeptide α-ketoamides have the followingstructural formula:

    M.sub.1 --AA--AA--AA--AA--CO--NR.sub.3 R.sub.4

or a pharmaceutically acceptable salt, wherein

M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂ --, X--NH--CO--, X₂N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂ N--SO₂ --, X--CO--,X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--;

X is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀fluoroalkyl, C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkylsubstituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substitutedwith K, phenyl disubstituted with K, phenyl trisubstituted with K,naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,naphthyl trisubstituted with K, C₁₋₁₀ alkyl with an attached phenylgroup, C₁₋₁₀ alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with anattached phenyl group substituted with K, C₁₋₁₀ alkyl with two attachedphenyl groups substituted with K, C₁₋₁₀ alkyl with an attached phenoxygroup, and C₁₋₁₀ alkyl with an attached phenoxy group substituted with Kon the phenoxy group;

J is selected from the group consisting of halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine, C₁₋₁₀alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--;

K is selected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfluoroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁₋₁₀ acyl, and C₁₋₁₀ alkoxy-CO--, andC₁₋₁₀ alkyl-S--;

AA is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ -CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine;

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₁₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ting attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, C₁₋₁₀ with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

The novel class of amino acid α-ketoamides have the following structuralformula:

    M.sub.1 AA--CO--NR.sub.2 R.sub.4

or a pharmaceutically acceptable salt, wherein

M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂ --, X--NH--CO--, X₂N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂ N--SO₂ --, X--CO--,X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--;

X is selected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀fluoroalkyl, C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkylsubstituted with J, 1-admantyl, 9-fluorenyl, phenyl, phenyl substitutedwith K, phenyl disubstituted with K, phenyl trisubstituted with K,naphthyl, naphthyl substituted with K, naphthyl disubstituted with K,naphthyl trisubstituted with K, C₁₋₁₀ alkyl with an attached phenylgroup, C₁₋₁₀ alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with anattached phenyl group substituted with K, C₁₋₁₀ alkyl with two attachedphenyl groups substituted with K, C₁₋₁₀ alkyl with an attached phenoxygroup, and C₁₋₁₀ alkyl with an attached phenoxy group substituted with Kon the phenoxy group;

J is selected from the group consisting of halogen, COOH, OH, CN, NO₂,NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine, C₁₋₁₀alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--;

K is selected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfloroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁₋₁₀ acyl, and C₁₋₁₀ alkoxy-CO--, andC₁₋₁₀ alkyl-S--;

AA is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glum mine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt2)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine;

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₃₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ring attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, Cl.10 with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

The following compounds are representatives of the invention:

Z--Leu--Phe--CONH--Et

Z--Leu--Phe--CONH--nPr

Z--Leu--Phe--CONH--nBu

Z--Leu--Phe--CONH--iBu

Z--Leu--Phe--CONH--Bzl

Z--Leu--Phe--CONH--(CH₂)₂ Ph

Z--Leu--Abu--CONH--Et

Z--Leu--Abu--CONH--nPr

Z--Leu--Abu--CONH--nBu

Z--Leu--Abu--CONH--iBu

Z--Leu--Abu--CONH--Bzl

Z--Leu--Abu--CONH--(CH₂)₂ Ph

Z--Leu--Abu--CONH--(CH₂)₃ --N(CH₂ CH₂)₂ O

Z--Leu--Abu--CONH--(CH₂)₇ CH₃

Z--Leu--Abu--CONH--(CH₂)₂ OH

Z--Leu--Abu--CONH--(CH₂)₂ O(CH₂)₂ OH

Z--Leu--Abu--CONH--(CH₂)₁₇ CH₃

Z--Leu--Abu--CONH--CH₂ --C₆ H₃ (OCH₃)₂

Z--Leu--Abu--CONH--CH₂ --C₄ H₄ N

Materials and Methods. HEPES, heparin, and A23187 were obtained fromCalbiochem. Suc--Leu--Tyr--AMC and chromogenic substrates were obtainedfrom Sigma. Calpain I was purified from human erythrocytes according tothe method of Kitahara (Kitahara et al., J. Biochem. 95, 1759-1766)omitting the Blue-Sepharose step. Calpain II from rabbit muscle andcathepsin B were purchased from Sigma. Papain was purchased fromCalbiochem.

Assay of Inhibitory Potency. Peptide α-ketoamides were assayed asreversible enzyme inhibitors. Various concentrations of inhibitors inMe₂ SO were added to the assay mixture which contained buffer andsubstrate. The reaction was started by the addition of the enzyme andthe hydrolysis rates were followed spectrophotometrically orfluorimetrically.

Calpain I from human erythrocytes and calpain II from rabbit wereassayed using Suc--Leu--Tyr--AMC [Sasaki et al., J. Biol. Chem. 259,12489-12494 (1984); incorporated herein by reference], and the AMC(7-amino-4-methylcoumarin) release was followed fluorimetrically(excitation at 380 nm, and emission at 460 nm). Calpains were assayed in25 mM Tris pH=8.0, 10 mM CaCl₂. Fluorescence was followed using a GilsonFL-1A fluorometer or a Perkin-Elmer 203 Fluorescence spectrometer.Cathepsin B was assayed in 20 mM sodium acetate pH=5.2, 0.5 mMdithiothreitol using Bz--Phe--Val--Arg--p--nitroanilide as substrate.Alternately, cathepsin B was assayed with Z--Arg--Arg--AFC [Barrett andKirschke, Methods Enzymol. 80, 535-561 (1981); incorporated herein byreference], and the AFC (7-amino-4-trifluoromethylcoumarin) release wasfollowed fluorimetrically (excitation at 400 nm and emission at 505 nm).Papain was assayed in 100 mM KPO₄, 1 mM EDTA, 2.5 mM cysteine pH=6.0using Bz--Arg--AMC or Bz--Arg--NA [Kanaoka et al., Chem. Pharm. Bull.25, 3126-3128 (1977); incorporated herein by reference] as a substrate.The AMC (7-amino-4-methylcoumarin) release was followed fluorimetrically(excitation at 380 nm, and emission at 460 nm). Enzymatic hydrolysisrates were measured at various substrate and inhibitor concentrations,and K_(I) values were determined by either Lineweaver-Burk plots orDixon plots.

A 0.1M Hepes, 0.5M NaCl, pH 7.5 buffer was utilized for human leukocyteelastase (HLE), porcine pancreatic elastase (PPE), chymotrypsin andcathepsin G. A 0.1 Hepes, 0.01M CaCl₂, pH 7.5 buffer was utilized fortrypsin, plasmin, and coagulation enzymes. A 50 mM Tris.HCl, 2 mM EDTA,5 mM cysteine, pH 7.5 was used as a buffer for papain. A 88 mM KH₂ PO4,12 mM Na₂ HPO₄, 1.33 mM EDTA, 2.7 mm cysteine, pH 6.0 solution was usedas a buffer for cathepsin B. A 20 mM Hepes, 10 mM CaCl₂, 10 mMmercatoethanol, pH 7.2 buffer was utilized for calpain I and calpain II.

HLE and PPE were assayed with MeO--Suc--Ala--Ala--Pro--Val--NA andSuc--Ala--Ala--Ala--NA, respectively [Nakajima et al., J. Biol. Chem.254, 4027-4032 (1979); incorporated herein by reference]. Humanleukocyte cathepsin G and chymotrypsin A.sub.α were assayed withSuc--Val--Pro--Phe--NA [Tanaka et al., Biochemistry 24, 2040-2047(1985); incorporated herein by reference]. The hydrolysis of peptide4-nitroanilides was measured at 410 nm [ε₄₁₀ =8800 M⁻¹ cm⁻¹ ; Erlangeret al., Arch. Biochem. Biophys. 95, pp 271-278 (1961); incorporatedherein by reference]. Trypsin, thrombin, human plasma kallikrein,porcine pancreatic kallikrein, human factor XIa, and human plasmin wereassayed with Z--Arg--SBzl or Z--Gly--Arg--SBu--i [McRae et al.,Biochemistry 20, 7196-7206 (1981); incorporated herein by reference].All peptide thioester hydrolysis rates were measured with assay mixturescontaining 4,4'-dithiodipyridine [ε₃₂₄ =19800 M⁻¹ cm⁻¹ ; Grasetti &Murray, Arch. Biochem. Biophys. 119, 41-49 (1967); incorporated hereinby reference].

Platelet membrane permeability assay. Calpain-mediated breakdown ofspectrin was measured by quantitative densitometry of thecalpain-specific 150/155 kDa spectrin fragment doublet [see Siman etal., Proc. Natl. Acad. Sci. USA 81, 3572-3576 (1984)]. Platelets wereisolated by a modification of the method of Ferrell and Martin [J. Biol.Chem. 264, 20723-20729 (1989)]. Blood (15-20 ml) was drawn from maleSprague-Dawley rats into 1/10th volume of 100 mM EDTA-citrate, andcentrifuged 10 minutes at 2000 rpm in a clinical centrifuge at roomtemperature. The plasma was resuspended in 15 ml of buffer 1 (136 mMNaCl, 2.7 mM KCl, 0.42 mM NaH₂ PO₄, 12 mM NaHCO₃, 2 mM MgCl₂, 2 mg/mlBSA (Sigma), 5.6 mM glucose, 22 nM Na₃ citrate pH 6.5) and plateletswere isolated at 2200 rpm at room temperature for 10 minutes. Plateletswere washed once in 15 ml buffer 1, then resuspended to 10⁷ cells/ml inbuffer 2 (136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH₂ PO₄, 12 mM NaHCO₃, 2 mMMgCl, 1 mg/ml BSA (Sigma), 5.6 mM glucose, 20 mM HEPES (Sigma) pH 7.4)and allowed to "rest" for a minimum of 10 minutes at room temperaturebefore use.

Inhibitors were added from stock solutions made fresh in DMSO. 100 μlplatelets, suspended to 10⁷ cells/ml in buffer 2, were incubated with 1μl of an inhibitor solution for 5 minutes at room temperature prior tothe addition of 2 mM Ca²⁺ and 1 uM A23187. After 10 minutes totalexposure to inhibitor (5 minutes exposure to ionophore) at roomtemperature, platelets were reisolated at 14,000 rpm for 10 sec in aBeckman microfuge, dissolved in SDS-PAGE sample buffer, and healed to90° C. for 3 minutes.

Samples were subjected to SDS-PAGE in 4-12% gradient mini gels (Novex)and transferred to nitrocellulose (Schleicher and Schuell 0.45 um) byelectroblotting. Filters were blocked for 10 minutes in 0.25% gelatin,1% BSA, 0.25% triton X₁₀₀, 0.9 % NaCl, 10 mM Tris-Cl pH 7.5, incubatedovernight in the same solution containing antibody to rat spectrin,washed 3×10 minutes with 10 mM Tris-Cl pH 7.5, 0.5% triton X 100,incubated 4 hours in wash buffer plus alkaline phosphatase conjugatedgoat anti-rabbit antibody (Biorad), and washed as above. Blots weredeveloped using the Biorad AP conjugate substrate kit. Quantitativedensitometry was used to obtain values for the intact spectrin bands andthe 150/155 kDa breakdown product doublet.

Structure-Activity Relationships. Table I shows the inhibition constants(K_(I)) for cathepsin B, calpain I, and calpain II. Dipeptideα-ketoamides with Abu and Phe in the P₁ site and Leu in the P₂ site arepotent inhibitors of calpain I and calpain II. Z--Leu--Abu--CONH--Et isa better inhibitor of calpain I than Z--Leu--Phe--CONH--Et by 14 fold.Replacement of the Z group (PhCH₂ OCO--) by similar groups such as PhCH₂CH₂ CO--, PhCH₂ CH₂ SO₂ --, PhCH₂ NHCO--, and PhCH₂ NHCS-- would alsoresult in good inhibitor structures. The best inhibitor of calpain II isZ--Leu--Abu--CONH--(CH₂)₂ --Ph. Changing the R₃ and R₄ groupssignificantly improves the inhibitory potency toward calpain II. Thebest dipeptide inhibitors are those which have long alkyl side chains(e.g. Z--Leu--Abu--CONH--(CH₂)₇ CH₃), alkyl side chains with phenylsubstituted on the alkyl group (e.g. Z--Leu--Abu--CONH--(CH₂)₂ --Ph), oralkyl groups with a morpholine ring substituted on the alkyl group [e.g.Z--Leu--Abu--CONH--(CH₂)₃ --Mpl, Mpl=--N(CH₂ CH₂)₂ O]. Dipeptideα-ketoamides with a small aliphatic amino acid residue or a Phe in theP₁ site are also good inhibitors for cathepsin B. The best inhibitor isZ--Leu--Abu--CONH--Et and replacement of the Z (PhCH₂ OCO--) by PhCH₂CH₂ CO--, PhCH₂ CH₂ SO₂ --, PhCH₂ NHCO--, and PhCH₂ NHCS-- would alsoresult in good inhibitor structures.

                  TABLE I                                                         ______________________________________                                        Inhibition of Cysteine Proteases by Peptide α-Ketoamides.                                   K.sub.I (uM)                                                                    Cal-   Cal-                                             Peptide α-Ketoamide                                                                           pain I pain II Cath B                                   ______________________________________                                        Z--Leu--Abu--CONH--Et 0.5    0.23    2.4                                      Z--Leu--Abu--CONH--nPr       0.25    8                                        Z--Leu--Abu--CONH--nBu                                                                              0.2            13                                       Z--Leu--Abu--CONH--iBu       0.14    4                                        Z--Leu--Abu--CONH--Bzl       0.35    2                                        Z--Leu--Abu--CONH--(CH.sub.2).sub.2 --Ph                                                                   0.022                                            Z--Leu--Abu--CONH--(CH.sub.2).sub.3 --Mpl                                                                  0.041                                            Z--Leu--Abu--CONH--(CH.sub.2).sub.7 CH.sub.3                                                               0.019                                            Z--Leu--Abu--CONH--(CH.sub.2).sub.17 CH.sub.3                                 Z--Leu--Abu--CONH--(CH.sub.2).sub.2 OH                                                                     0.078                                            Z--Leu--Abu--CONH--(CH.sub.2).sub.2 O(CH.sub.2).sub.2 OH                                            0.16                                                    Z--Leu--Phe--CONH--Et 7.0    0.32    6                                        Z--Leu--Phe--CONH--nPr                                                                              15.0   0.05    3                                        Z--Leu--Phe--CONH--nBu       0.028   3                                        Z--Leu--Phe--CONH--iBu       0.065   4                                        Z--Leu--Phe--CONH--Bzl       0.046                                            Z--Leu--Phe--CONH(CH.sub.2).sub.2 Ph                                                                       0.024                                            ______________________________________                                    

Table II shows the inhibition constants (K_(I)) for PP elastase andchymotrpysin. Dipeptide α-ketoamides with Abu in the P₁ site are potentinhibitors of PP elastase. The structures with medium sizedstraight-chain alkyl groups such as n--Pr and n--Bu were betterinhibitors than a small alkyl (Et) or a branched alkyl (i--Bu).Dipeptide α-ketoamides with Phe in the P₁ site are moderate inhibitorsof chymotrypsin. The inhibitor with R₃ =n--Bu and R₄ =H was the best inthe series. In general the inhibitors were more potent at inhibitingcysteine protease than serine proteases. Extending the peptide chain totripeptide or tetrapeptide ketoamides would improve the inhibitorypotency toward serine proteases.

                  TABLE II                                                        ______________________________________                                        Inhibition of Serine Proteases by Peptide α-Ketoamides.                                      K.sub.I (uM)                                                                    Chymo-                                                 Peptide α-Ketoamide                                                                            trypsin PP elastase                                    ______________________________________                                        Z--Leu--Abu--CONH--Et  >150    65                                             Z--Leu--Abu--CONH--nPr >300    2                                              Z--Leu--Abu--CONH--nBu >300    5                                              Z--Leu--Abu--CONH--iBu >300    40                                             Z--Leu--Abu--CONH--Bzl >300                                                   Z--Leu--Abu--CONH--(CH.sub.2).sub.2 --Ph                                      Z--Leu--Abu--CONH--(CH.sub.2).sub.3 --Mpl                                     Z--Leu--Abu--CONH--(CH.sub.2).sub.7 CH.sub.3                                  Z--Leu--Abu--CONH--(CH.sub.2).sub.17 CH.sub.3                                 Z--Leu--Abu--CONH--(CH.sub.2).sub.2 OH                                        Z--Leu--Abu--CONH--(CH.sub.2).sub.2 O(CH.sub.2).sub.2 OH                      Z--Leu--Phe--CONH--Et  73      >150                                           Z--Leu--Phe--CONH--nPr 18      >300                                           Z--Leu--Phe--CONH--nBu 8       >100                                           Z--Leu--Phe--CONH--iBu 24                                                     Z--Leu--Phe--CONH--Bzl                                                        Z--Leu--Phe--CONH(CH.sub.2).sub.2 Ph                                          ______________________________________                                    

Peptide α-ketoamide were substantially more stable in both plasma andliver than the corresponding peptide α-ketoesters (Table III). Thepeptide α-ketoamides were also much more effective in the plateletassay. Extending the R₃ group to an alkyl group or an alkyl groupsubstituted with a phenyl group increased the membrane permeability ofthe inhibitors as indicated by increased potency in the platelet assay.

                  TABLE III                                                       ______________________________________                                        Half-lives Plasma and in Liver and Activity in the Platelet Assay.                                         t.sub.1/2                                                                              t.sub.1/2                               Peptide α-Ketoamide or Ester                                                                platelet plasma   liver                                   ______________________________________                                        Z--Leu--Abu--COOEt  42       2.8                                              Z--Leu--Abu--COOn--Bu                                                                             28                                                        Z--Leu--Abu--COOBzl ++                                                        Z--Leu--Leu--Abu--COOEt                                                                           40                                                        2-NapSO2--Leu--Leu--Abu--COOEt                                                                    100      >60                                              2-NapCO--Leu--Leu--Abu--COOEt                                                                              25                                               Tos--Leu--Leu--Abu--COOEt                                                                         30       30                                               Z--Leu--Abu--COOH   8        >60      >60                                     Z--Leu--Abu--CONH--Et                                                                             1.5      >60      >60                                     Z--Leu--Abu--CONH--nPr                                                                            70       >60      >60                                     Z--Leu--Abu--CONH--nBu                                                                            2.0      >60      >60                                     Z--Leu--Abu--CONH--iBu                                                                            28       >60                                              Z--Leu--Abu--CONH--Bzl                                                                            1.5      >60      >60                                     Z--Leu--Phe--COOEt  42       7.8                                              Z--Leu--Phe--COOnBu +++      7.7                                              Z--Leu--Phe--COOBz  ++       1.9                                              Z--Leu--Leu--Phe--COOEt                                                                           ++                                                        Z--Leu--Phe--COOH   6.5      >60      >60                                     Z--Leu--Phe--CONH--Et                                                                             1.7      >60      >60                                     Z--Leu--Phe--CONH--nPr                                                                            24       >60      >60                                     Z--Leu--Phe--CONH--nBu                                                                            38       >60      >60                                     Z--Leu--Phe--CONH--iBu                                                                            22       >60                                              Z--Leu--Phe--CONH--Bzl                                                        Z--Leu--Phe--CONH(CH.sub.2).sub.2 Ph                                                              3.0      >60                                              Z--Leu--Nle--COOEt  20       3.7                                              Z--Leu--Nva--COOEt  40       2.8                                              Z--Leu--Met--COOEt  +        8                                                ______________________________________                                         (+++ = excellent activity; ++ = good activity, + = moderate activity;         quantitative measurements not yet complete)                              

Inhibition Mechanism. A crystal structure of one α-ketoester bound intothe active site of porcine pancreatic elastase has been completed and aschematic drawing of the interactions observed is shown below. Theactive site Ser-195 oxygen of the enzyme has added to the carbonyl groupof the ketoester to form a tetrahedral intermediate which is stabilizedby interactions with the oxyanion hole. This structure resembles thetetrahedral intermediate involved in peptide bond hydrolysis and provesthat α-ketoesters are transition-state analogs. His-57 is hydrogenbonded to the carbonyl group of the ester functional group, the peptidebackbone on a section of PPE's backbone hydrogen bonds to the inhibitorto form a β-sheet, and the benzyl ester is directed toward the S'subsites. The side chain of the P₁ amino acid residue is located in theS₁ pocket of the enzyme. Interactions with ketoamides would be similarexcept for that there would be the possibility of forming an additionalhydrogen bond with the NH group of the ketoamide functional group if R₃or R₄ was H. If R₃ and/or R₄ are longer substituents, then they wouldmake favorable interactions with the S' subsites of the enzyme. ##STR1##

The active site of cysteine proteases share several features in commonwith serine proteases including an active site histidine residue. Inplace of the Ser-195, cysteine proteases have an active site cysteineresidue which would add to the ketonic carbonyl group of the peptideketo acids, keto esters, or ketoamides to form an adduct very similar tothe structure depicted above except with a cysteine residue replacingthe serine-195 residue. Additional interactions would occur between theextended substrate binding site of the cysteine protease and theinhibitor which would increase the binding affinity and specificity ofthe inhibitors.

Inhibitor Design and Selection. The peptide and amino acid α-ketoamidederivatives, as shown in the above crystal structure, bind to theenzymes using many of the interactions that are found in complexes of aparticular individual enzyme with its substrates. In order to design aninhibitor for a particular serine or cysteine protease, it is necessaryto: 1) find the amino acid sequences of good peptide substrates for thatenzyme, and 2) place those or similar amino acid sequences into aα-ketoamide structure. Additional interactions with the enzyme can beobtained by tailoring the R group of the inhibitor to imitate the aminoacid residues which are preferred by an individual protease at the S₁ 'and S₂ ' subsites. For example, ketoesters with R₃ and/or R₄ =branchedalkyl groups would interact effectively with serine and cysteineproteases which prefer Leu, Ile, and Val residues at P₁ ' and/or P₂ ',while amides with R=alkyl substituted with phenyl would interacteffectively with serine and cysteine proteases which prefer Phe, Tyr,Trp residues at P₁ ' and/or P₂ '. Likewise, the M₁ group can be tailoredto interact with the S subsites of the enzyme. This design strategy willalso work when other classes of peptide inhibitors are used in place ofthe peptide substrate to gain information on the appropriate sequence toplace in the ketoester, ketoacid, or ketoamide inhibitor. Thus, we areable to predict the structure of new inhibitors for other serine andcysteine proteases based on knowledge of their substrate specificities.Once a good inhibitor structure for a particular enzyme is found, it isthen possible to change other characteristics such as solubility orhydrophobicity by adding substituents to the M₁ or R₃ and R₄ groups.

Elastase is an enzyme which hydrolyzes most effectively tetra- andtripeptides having P₁ residues with small alkyl side chains such as Alaand Val. MeO--Suc--Ala--Ala--Ala--Val--NA and Z--Ala--Ala--Ala--Ala--NAare good substrates (NA=4-nilroanilide). Thus the correspondingα-ketoamide Z--Ala--Ala--Ala--DL--Ala--CO--NR₃ R₄ andMeO--Suc--Ala--Ala--Pro--DL--Abu--CO--NR₃ R₄ will be excellent elastaseinhibitors. Suc--Phe--Leu--Phe--NA is an excellent substrate forchymotrypsin, cathepsin G, and mast cell chymases. Thus, thecorresponding α-ketoamide will be an excellent inhibitor for thesechymotrypsin-like enzymes. In the case of the cysteine protease calpain,a good inhibitor sequence is Ac--Leu--Leu--Nle--H. We have found thatketoesters related in structure such as Z--Leu--Abu--CO--NR₃ R₄ andZ--Leu--Phe--CO--NR₃ R₄ are potent inhibitors for calpain.

The following structures are predicted to be potent inhibitors for thelisted enzymes. The inhibitor sequences were obtained from peptidesubstrate and/or inhibitor sequences in the protease literature.

    __________________________________________________________________________    Z--Gly--Leu--Phe--CO--NR.sub.3 R.sub.4                                                                for cathepsin G and RMCP II                           MeO--Suc--Ala--Ala--Pro--Met--CO--NR.sub.3 R.sub.4                                                    for cathepsin G                                       Boc--Ala--Ala--Asp--CO--NR.sub.3 R.sub.4                                                              for human lymphocyte granzyme B                       Suc--Pro--Leu--Phe--CO--NR.sub.3 R.sub.4 and Boc--Ala--Ala--Phe--CO--NR.su    b.3 R.sub.4                                                                                           for RMCP I (RMCP = rat mast cell protease)            Boc--Gly--Leu--Phe--CO--NR.sub.3 R.sub.4, Suc--Phe--Leu--Phe--CO--NR.sub.3     R.sub.4                                                                                              for human and dog skin chymase                        Boc--Ala--Ala--Glu--CO--NR.sub.3 R.sub.4                                                              for S. aureus V-8 protease                            Z--Gly--Gly--Pro--CO--NR.sub.3 R.sub.4                                                                for human prolyl endopeptidase                        Ala--Pro--CO--NR.sub.3 R.sub.4                                                                        for DPP IV                                            Suc--Ala--Ala--Pro--Val--CO--NR.sub.3 R.sub.4                                                         for PPE                                               Suc--Lys(Cbz)--Val--Pro--Val--CO--NR.sub.3 R.sub.4, adamantyl-SO.sub.2        --Lys(COCH.sub.2 CH.sub.2 CO.sub.2 H)--Ala--Val--                             CO--NR.sub.3 R.sub.4, adamantyl--CH.sub.2 CH.sub.2 OCO--Glu(O--t-Bu)--Pro-    -Val--CO--NR.sub.3 R.sub.4, and adamantyl-                                    SO.sub.2--Lys(CO--C.sub.6 H.sub.4 CO.sub.2 H)--Ala--Val--CO--NR.sub.3         R.sub.4                 for human leukocyte(neutrophil) elastase              Suc--Ala--Ala--Pro--Leu--CO--NR.sub.3 R.sub.4                                                         for elastolytic proteinase from "Schistosoma                                  mansoni"                                              Glu--Phe--Lys--CO--NR.sub.3 R.sub.4 and Dns--Ala--Phe--Lys--CO--NR.sub.3      R.sub.4                                                                                               for plasmin                                           D--Val--Gly--Arg--CO--NR.sub.3 R.sub.4 and Dns--Glu--Gly--Arg--CO--NR.sub.    3 R.sub.4                                                                                             for factor Xa                                         Z--Phe--Arg--CO--NR.sub.3 R.sub.4 and Z--Trp--Arg--CO--NR.sub.3 R.sub.4                               for porcine pancreatic and human plasma                                       kallikreins                                           Z--Lys--Arg--CO--NR.sub.3 R.sub.4                                                                     for human skin tryptase                               Z--Gly--Arg--CO--NR.sub.3 R.sub.4                                                                     for human lung tryptase                               Z--Ile--Ala--Gly--Arg--CO--NR.sub.3 R.sub.4                                                           for factors IXa, Xa, XIa, XIIa and                                            bovine plasma kallikrein                              Glu--Gly--Arg--CO--NR.sub.3 R.sub.4                                                                   for urokinase                                         Dns--Phe--Pro--Arg--CO--NR.sub.3 R.sub.4                                                              for plasminogen activator                             Dns--Ile--Pro--Arg--CO--NR.sub.3 R.sub.4                                                              for activated protein C                               Z--Trp--Arg--CO--NR.sub.3 R.sub.4                                                                     for bovine factor IXa                                 Z--Gly--Arg--CO--NR.sub.3 R.sub.4                                                                     for bovine factor Xa and XIa                          Z--Phe--Arg--CO--NR.sub.3 R.sub.4                                                                     for bovine factor XIIa                                Dns--Glu--Gly--Arg--CO--NR.sub.3 R.sub.4                                                              for human factor Xa                                   D--Phe--Pro--Arg--CO--NR.sub.3 R.sub.4, D--MePhe--Pro--Arg--CO--NR.sub.3      R.sub.4 , and                                                                 Boc--D--Phe--Pro--Arg--CO--NR.sub.3 R.sub.4                                                           for human thrombin                                    Z--Phe--Gly--Arg--CO--NR.sub.3 R.sub.4                                                                for trypsin                                           Cl--C.sub.6 H.sub.4 CH.sub.2 OCO--Phe--Gly--CO--NR.sub.3 R.sub.4                                      for papain                                            C.sub.6 H.sub.5 CH.sub.2 NHCO--Gly--Phe--Gly--CO--NR.sub.3 R.sub.4                                    for cathepsin B                                       __________________________________________________________________________

R₃ and R₄ are selected independently from the group consisting of H,C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl, C₁₋₂₀ alkyl with a phenyl groupattached to the C₁₋₂₀ alkyl, C₃₋₂₀ cyclized alkyl with an attachedphenyl group, C₁₋₂₀ alkyl with an attached phenyl group substituted withK, C₁₋₂₀ alkyl with an attached phenyl group disubstituted with K, C₁₋₂₀alkyl with an attached phenyl group trisubstituted with K, C₃₋₂₀cyclized alkyl with an attached phenyl group substituted with K, C₁₋₁₀alkyl with a morpholine [--N(CH₂ CH₂)O] ring attached through nitrogento the alkyl, C₁₋₁₀ alkyl with a piperidine ring attached throughnitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidine ring attachedthrough nitrogen to the alkyl, C₁₋₂₀ alkyl with an OH group attached tothe alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ with an attached 4-pyridylgroup, C₁₋₁₀ with an attached 3-pyridyl group, C₁₋₁₀ with an attached2-pyridyl group, C₁₋₁₀ with an attached cyclohexyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).

In Vitro Uses. To use the above inhibitors in vitro, they are dissolvedin an organic solvent such as dimethylsulfoxide or ethanol, and areadded to an aqueous solution containing seine and/or cysteine proteases.The final concentration of the organic solvent should be less than 25%.The inhibitors may also be added as solids or in suspension. The serineand cysteine protease inhibitors of this invention would be useful in avariety of experimental procedures where proteolysis is a significantproblem. Inclusion of these inhibitors in a radioimmunoassay experimentswould result in higher sensitivity. The use of these inhibitors inplasma fractionation procedures would result in higher yields ofvaluable plasma proteins and would make purification of the proteinseasier. The inhibitors disclosed here could be used in cloningexperiments utilizing bacterial cultures, yeast and human cells to yielda purified cloned product in higher yield.

The novel compounds of this invention are effective in the prevention ofunnecessary proteolysis caused by chymotrypsin-like and elastase-likeenzymes in the process of purification, transport and storage ofpeptides and proteins as shown in Table II by effective inhibition ofchymotrypsin and elastase and other cysteine proteases.

In Vivo Uses. Effective inhibitors of the proteolytic function of humanleukocyte elastase and chymotrypsin-like enzymes (Table II) would haveanti-inflammatory activity and can be used to treat and controlemphysema, adult respiratory distress syndrome and rheumatoid arthritis.Effective inhibitors of the proteolytic function of chymotrypsin andpancreatic elastase (Table ll) are effective for therapeutic use in thetreatment of pancreatitis.

Peptide α-ketoamide can be used to control protein turnover, musculardystrophy, myocardial tissue damage, tumor metastasis, and boneresorption as shown in Table I by effective inhibition of lysosomalcathepsin B in buffer. Peptide α-ketoamides can also be used asneuroprotectants or for the treatment of ischemia, stroke or Alzheimer'sdisease as shown in Table I by effective inhibition of calpain I andcalpain II.

Considerable evidence has shown that leukocyte elastase and/or relatedenzymes play a role in tumor cell metastasis [Salo et al., Int. J.Cancer 30, pp 669-673 (1973); Kao et al., Biochem. Biophys. Res. Comm.105, pp 383-389 (1982); Powers, J. C. in Modification of Proteins, R. E.Feeney and J. R. Whitaker, eds., Adv. Chem. Ser 198, Amer. Chem. Soc.,Wash., D.C. pp 347-367 (1982); all incorporated herein by reference],therefore it is suggested that compounds of this invention may haveanti-tumor activity.

Pulmonary emphysema is a disease characterized by progressive loss oflung elasticity due to the destruction of lung elastin and alveoli. Thedestructive changes of lung parentchyma associated with pulmonaryemphysema are caused by uncontrolled proteolysis in lung tissues[Janoff, Chest 83, 54-58 (1983); incorporated herein by reference]. Anumber of proteases have been shown to induce emphysema in animals[Marco et al., Am. Rev. Respir. Dis. 104, 595-598 (1971); Kaplan, J.Lab. Clin. Med. 82, 349-356 (1973); incorporated herein by reference],particularly human leukocyte elastase [Janoff, ibid 115, 461-478 (1977);incorporated herein by reference]. Leukocyte elastase and othermediators of inflammation also appear to play a role in diseases such asmucocutaneous lymph node syndrome [Reiger et al., Eur. J. Pediatr. 140,92-97 (1983); incorporated herein by reference] and adult respiratorydistress syndrome [Stockley, Clinical Science 64, 119-126 (1983); Lee etal., N. Eng. J. Med. 304, 192-196 (I981); Rinaldo, ibid 301, 900-909(1982); incorporated herein by reference].

It is known that in vitro activity of elastase inhibitors correlateswith in vivo activity in animal models of emphysema and inflammation[Otterness et al., editors, Advances in Inflammation Research, Vol. 11,Raven Press 1986; incorporated herein by reference]. Prophylacticadministration of an inhibitor of elastase significantly diminishes theextent of elastase-induced emphysema [Kleinerman et al., Am. Rev. Resir.Dis. 121, 381-387 (1980); Lucey et . al., Eur. Respir. J. 2, 421-427(1989); incorporated herein by reference]. Thus the novel inhibitorsdescribed here should be useful for the treatment of emphysema andinflammation. Elastase inhibitors have been used orally, by injection,or by instillation in the lungs in animal studies (Powers, Am. Rev.Respir. Dis., 127, s54-s58 (1983); Powers and Bengali, Am. Rev. Respir.Dis. 134, 1097-1100 (1986); these two articles are incorporated hereinby reference). The inhibitors described above can be used by any ofthese routes.

Drug Delivery. For therapeutic use, the peptide α-ketoamides may beadministered orally, topically or parenterally. The term parenteral asused includes subcutaneous injection, intravenous, intramuscular,intrasternal injection or infusion techniques. The dosage dependsprimarily on the specific formulation and on the object of the therapyor prophylaxis. The amount of the individual doses as well as theadministration is best determined by individually assessing theparticular case.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules or syrups or elixirs. Dosage levels ofthe order to 0.2 mg to 140 mg per kilogram of body weight per day areuseful in the treatment of above-indicated conditions (10 mg to 7 gmsper patient per day). The amount of active ingredient that may becombined with carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration.

For injection, the therapeutic amount of the peptide α-ketoamides ortheir pharmaceutically acceptable salts will normally be in the dosagerange from 0.2 to 140 mg/kg of body weight. Administration is made byintravenous, intramuscular or subscutaneous injection. Accordingly,pharmaceutical compositions for parenteral administration will containin a single dosage form about 10 mg to 7 gms of the compounds per dose.In addition to the active ingredient, these pharmaceutical compositionswill usually contain a buffer, e.g. a phosphate buffer which keeps thepH in the range from 3.5 to 7 and also sodium chloride, mannitol orsorbitol for adjusting the isotonic pressure.

A composition for topical application can be formulated as an aqueoussolution, lotion, jelly or an oily solution or suspension. A compositionin the form of an aqueous solution is obtained by dissolving thecompounds of this invention in aqueous buffer solution of pH 4 to 6.5and if desired, adding a polymeric binder. An oily formulation fortopical application is obtained by suspending the compounds of thisinvention in an oil, optionally with the addition of a swelling agentsuch as aluminium stearate and/or a surfactant.

SYNTHETIC METHODS

The α-ketoamide inhibitors are prepared from the correspondingα-ketoaamide. The ketoester inhibitors are prepared by a two stepDakin-West from the corresponding peptide acid (Charles et al., J. Chem.Soc. Perkin 1, 1139-1146, 1980). This procedure can be utilized witheither amino acid derivatives, dipeptide derivatives, tripeptidederivatives, or tetrapeptide derivatives as shown in the followingscheme.

    M.sub.1 --AA--OH→Enol Ester→M.sub.1 --AA--CO--O--R

    M.sub.1 --AA--AA--OH→Enol Ester→M.sub.1 --AA--AA--CO--O--R

    M.sub.1 --AA--AA--AA--OH→Enol Ester→M.sub.1 --AA--AA--AA--CO--O--R

    M.sub.1 --AA--AA--AA--AA--OH→Enol Ester→M.sub.1 --AA--AA--AA--AA--CO--O--R

The precursor peptide can be prepared using standard peptide chemistrywhich is well described in publications such as The Peptides, Analysis,Synthesis, Biology, Vol. 1-9, published in 1979-1987 by Academic Pressand Houben-Weyl Methoden der Organischen Chemie, Vol. 15, Parts 1 and 2,Synthese von Peptiden, published by Georg Thieme Verlag, Stuttgart in1974 (both references incorporated herein by reference).

The M₁ group can be introduced using a number of different reactionschemes. First it could be introduced directly on an amino acid as shownin the following scheme (top), or the M₁ group could be introduced byreaction with an amino acid ester, followed by removal of the estergroup to give the same product (bottom).

    H--AA--OH→M.sub.1 --AA--OH

    H--AA--O R'→M.sub.1 --AA--O R'→M.sub.1 --AA--OH

The techniques for introduction of the M₁ group is well documented inthe The Peptides, Houben-Weyel, and many other textbooks on organicsynthesis. For example reaction with cyanate or p-nitrophenyl cyanatewould introduce a carbamyl group (M₁ =NH₂ CO--). Reaction with Me₂ NCOClwould introduce the Me₂ NCO-group. Reaction with p-nitrophenylthiocarbamate would introduce a thio carbamyl group (M₁ --NH₂ CS--).Reaction with NH₂ SO₂ Cl would introduce the NH₂ SO₂ -group. Reactionwith Me₂ NSO₂ Cl would introduce the Me₂ NSO₂ -group. Reaction with asubstituted alkyl or aryl isocyanate would introduce the X--NH--CO-groupwhere X is a substituted alkyl or aryl group. Reaction with asubstituted alkyl or aryl isothiocyanate would introduce theX--NH--CS---group where X is a substituted alkyl or aryl group. Reactionwith X--SO₂ --Cl would introduce the X--SO₂ -group. Reaction with asubstituted alkyl or aryl acid chloride would introduce an acyl group(M=X--CO--). For example, reaction with MeO--CO--CH₂ CH₂ --CO--Cl wouldgive the X--CO-group where X is a C₂ alkyl substituted with a C₁alkyl-OCO-group. Reaction with a substituted alkyl or aryl thioacidchloride would introduce a thioacyl group (M=X--CS--). Reaction with ana substituted alkyl or aryl sulfonyl chloride would introduce an X--SO₂-group. For example reaction with clansyl chloride would give the X--SO₂-derivative where X was a naphthyl group mono substituted with adimethylamino group. Reaction with a substituted alkyl or arylchloroformate would introduce a X--O--CO-group. Reaction with asubstituted alkyl or aryl chlorothioformate would introduce aX--O--CS--. There are many alternate reaction schemes which could beused to introduce all of the above M₁ groups to give either M₁ --AA--OHor M₁ --AA--OR¹.

The M₁ --AA--OH derivatives could then be used directly in theDakin-West reaction or could be converted into the dipeptides,tripeptides, and tetrapeptides M₁ --AA--AA--OH, M₁ --AA--AA--AA--OH, orM₁ --AA--AA--AA--AA--OH which could be used in the Dakin-West reaction.The substituted peptides M₁ --AA--AA--OH, M₁ --AA--AA--AA--OH, or M₁--AA--AA--AA--AA--OH could also be prepared directly from H--AA--AA--OH,H--AA--AA--AA--OH, or H--AA--AA--AA--AA--OH using the reactionsdescribed above for introduction of the M₁ group. Alternately, the M₁group could be introduced by reaction with carboxyl blocked peptides togive M₁ --AA--AA--OR', M₁ --AA--AA--AA--OR', or M₁--AA--AA--AA--AA--OR', followed by the removal of the blocking group R'.

The R group in the ketoester structures is introduced during theDakin-West reaction by reaction with an oxalyl chlorideCl--CO--CO--O--R. For example, reaction of M₁ --AA--AA--OH with ethyloxalyl chloride C₁ --CO--CO--O--Et gives the keto ester M₁--AA--AA--CO--O--Et. Reaction of M₁ --AA--AA--AA--AA--OH withCl--CO--CO--O--Bzl would give the ketoester M₁--AA--AA--AA--AA--CO--O--Bzl. Clearly a wide variety of R groups can beintroduced into the ketoester structure by reaction with various alkylor arylalkyl oxalyl chlorides (Cl--CO--CO--O--R). The oxalyl chloridesare easily prepared by reaction of an alkyl or arylalkyl alcohol withoxalyl chloride Cl--CO--CO--Cl. For example, Bzl--O--CO--CO--Cl andn--Bu--O--CO--CO--Cl are prepared by reaction of respectively benzylalcohol and butanol with oxalyl chloride in yields of 50% and 80%[Warren, C. B., and Malee, E. J., J. Chromatography 64, 219-222 (1972);incorporated herein by reference].

Ketoamides M₁ --AA--CO--NR₃ R₄, M--AA--AA--CO--NR₃ R₄,M--AA--AA--AA--CO--NR₃ R₄, M--AA--AA--AA--AA--CO--NR₃ R₄ were preparedindirectly from the ketoesters. The ketone carbonyl group was firstprotected as shown in the following scheme and then the ketoamide wasprepared by reaction with an amine H--NR₃ R₄. The illustrated procedureshould also work with other protecting groups. In addition, thecorresponding ketoacid could be used as a precursor. Blocking the ketonecarbonyl group of the ketoacid and then coupling with an amine H--NR₃ R₄using standard peptide coupling reagents would yield an intermediatewhich could then be deblocked to form the ketoamide. ##STR2##

The following detailed examples are given to illustrate the inventionand are not intended to limit it in any mariner.

EXAMPLE 1

Z--Leu--Phe--CONH--Et

To a stirred solution of Z--Leu--Phe--OH (20 g, 48.5 mmole),4-dimethylaminopyridine (0.587 g, 4.8 mmole), and pyridine (15.7 ml, 194mmole) in anhydrous THF (100 ml) was added ethyl oxalyl chloride (11.4ml, 101.8 mmole) at a rate sufficient to initiate refluxing. The mixturewas gently refluxed for 4 hours, cooled to room temperature, and water(80 ml) was added. The reaction mixture was stirred vigorously for 30min, and extracted with ethyl acetate (3×100 ml). The combined organiclayers were washed with water (2×100 ml), saturated sodium chloride(2×100 ml), decolorized with decolorizing carbon, dried over magnesiumsulfate, and concentrated, leaving a dark orange oil. Chromatography ona silica gel column with CHCl₃ /CH₃ OH (50:1 v/v) afforded 14.63 g(y=53%) of Z--Leu--Phe--enolester. The product was a yellow oil. Singlespot on TLC, R_(f) =0.77 (CHCL₃ /CH₃ OH 50:1 ). NMR (CDCl₃) ok.

To a stirred pale yellow solution of the Z--Leu--Phe--enolester (14.63g, 25.73 mmole) in anhydrous ethanol (50 ml) was added a solution ofsodium ethoxide (0.177 g, 2.6 mmole) in ethanol (5 ml). The orangesolution was stirred for 3 hours at room temperature, then the ethanolwas evaporated and the residue was treated with ethyl ether (300 ml).The ether layer was washed with water (2×100 ml), saturated sodiumchloride (2×100 ml), dried over magnesium sulfate, and concentrated,leaving a orange oil. Chromatography on a silica gel column with CHCl₃/CH₃ OH (50:1 v/v) afforded 7.76 g (y=64%) of the α-ketoesterZ--Leu--Phe--COOEt. The product was a yellow oil. Single spot on TLC,R_(f) =0.44 (CHCl₃ /CH₃ OH 50:1). NMR (CDCl₃) ok. MS (FAB, calcd. forC₂₆ H₃₂ N₂ O₆ : 468.6), m/e=469 (M+1).

The α-carbonyl group of Z--Leu--Phe--COOEt was protected by thefollowing procedure. To a solution of Z--Leu--Phe--COOEt (1 g, 2.13mmole) in 5 ml of CH₂ Cl₂ was added 1,2-ethanedithiol (0.214 ml, 2.55mmole), followed by 0.5 ml of boron trifluoride etherate. The solutionwas stirred overnight at room temperature. Water (20 ml) and ethyl ether(20 ml) were added. The organic layer was separated, washed with water(2×10 ml), saturated sodium chloride (2×10 ml), dried over magnesiumsulfate, and evaporated to afford 0.98 g (y=84%) yellow semisolid.

The protected α-ketoester (0.98 g, 1.8 mmole) was dissolved in ethanol(5 ml), cooled to 0°-5° C. in an ice bath, and ethylmine was bubbledthrough the solution until 2.43 g (54 mmole) had been added. Thereaction mixture was allowed to warm to room temperature slowly, andstirred overnight. The mixture was filtered, a white precipitate wasremoved, leaving a yellow semisolid. Chromatography on a silica gelcolumn with CHCl₃ /CH₃ OH (30:1 v/v) afford 0.63 g (y=75%) ofZ--Leu--Phe--CONH--Et. The product was a pale yellow solid. Single spoton TLC, R_(f) =0.60 (CHCl₃ /CH₃ OH 20:1); mp 145°-147° C. Anal. calcd.for C₂₆ H₃₃ N₃ O₅ : 467.56; C, 66.79; H, 7.11; N, 8.99; found: C, 66.59;H, 7.09; N, 8.95. NMR (CDCl₃) ok. MS (FAB) m/e=468 (M+1).

EXAMPLE 2

Z--Leu--Phe--CONH--nPr

This compound was synthesized from the protected α-ketoester andpropylamine in 92% yield by the procedure described in Example 1. Singlespot on TLC, R_(f) =0.50 (CHCl₃ /CH₃ OH 50:1); mp 152°-153 ° C. Anal.calcd. for C₂₇ H₃₅ N₃ O₅ : 481.57; C, 67.33; H, 7.33; N, 8.72. Found: C,67.21; H, 7.38; N, 8.64. NMR (CDCl3) ok. MS (FAB) m/e=482 (M+1).

EXAMPLE 3

Z--Leu--Phe--CONH--nBu

This compound was synthesized from the protected α-ketoester andbutylamine in 67% yield by the procedure described in Example 1. Singlespot on TLC, R_(f) =0.50. (CHCl₃ /CH₃ OH 50:1); mp 152°-153 ° C. Anal.calcd. for C₂₈ H₃₇ N₃ O₅ : 495.59; C, 67.85; H, 7.52; N, 8.48. Found: C,67.70; H, 7.57; N, 8.43. NMR (CDCl₃) ok. MS (FAB) m/e=496 (M+1).

EXAMPLE 4

Z--Leu--Phe--CONH--iBu

This compound was synthesized from the protected α-ketoester andisobutylamine in 53% yield by the procedure described in Example 1.Single spot on TLC, R_(f) =0.54 (CHCl₃ /CH₃ OH 50:1); mp 152° C. Anal.calcd. for C₂₈ H₃₇ N₃ O₅ : 495.59; C, 67.85; H, 7.52; N, 8.48. Found: C,67.77; H, 7.56; N, 8.40. NMR (CDCl₃) ok. MS (FAB) me/=496 (M+1).

EXAMPLE 5

Z--Leu--Phe--CONH--Bzl

This compound was synthesized from the protected α-ketoester andbenzylamine in 40% yield by the procedure described in Example 1. Afterreacting overnight, ethyl acetate (60 ml) was added. The mixture wasfiltered to remove a white precipitate. The solution was washed withcooled 1N HCl (3×25 ml), water (1×20 ml), saturated sodium chloride(2×20 ml), and dried over magnesium sulfate. The solution was evaporatedleaving a yellow solid. Chromatography on a silica gel column with CHCl₃/CH₃ OH 30:1 v/v) afforded a yellow solid. Single spot on TLC, R_(f)=0.45 (CHCl₃ /CH₃ OH 30:1); mp 160°-162° C. Anal. calcd. for C₃₁ H₃₅ N₃O₅ : 529.61; C, 70.30; H, 6.66; N, 7.93. Found: C, 70.18; H, 6.67; N,7.99. NMR (CDCl3) ok. MS (FAB) m/e=530 (M+1).

EXAMPLE 6

Z--Leu--Phe--CONH--(CH₂)₂ Ph

This compound was synthesized from the protected α-ketoester andphenethylamine in 50% yield by the procedure described in Example 5.Single spot on TLC, R_(f) =0.50 (CHCl₃ /CH₃ OH 30:1); mp 151°-153° C.Anal. calcd. for C₃₂ H₃₇ N₃ O₅ : 543.66; C, 70.70; H, 6.86; N, 7.73.Found: C, 70.54; H, 6.88; N, 7.74. NMR (CDCl₃) ok. MS (FAB) m/e=544(M+1).

EXAMPLE 7

Z--Leu--Abu--CONH--Et

This compound was synthesized from protected α-ketoester derived fromZ--Leu--Abu--CO₂ Et and ethylamine in 64% yield by the proceduredescribed in Example 1. Single spot on TLC, R_(f) =0.36 (CHCl₃ /CH₃ OH50;1); mp 130°-132° C. Anal. calcd. for C₂₁ H₃₁ N₃ O₅ : 405.45; C,62.20; H, 7.71; N, 10.36. Found: C, 61.92; H, 7.62; N, 10.31. NMR(CDCl₃) ok. MS (FAB) m/e=406 (M+1).

EXAMPLE 8

Z--Leu--Abu--CONH--nPr

This compound was synthesized from the corresponding protectedα-ketoester and propylamine in 47% yield by the procedure described inExample 1. Single spot on TLC, R_(f) =0.28 (CHCl₃ /CH₃ OH 50:1); mp134°-135° C. Anal. calcd. for C₂₂ H₃₃ N₃ O₅ : 419.50; C, 62.98; H, 7.93;N, 10.02. Found: C, 62.84; H, 7.97; N, 9.94. NMR (CDCl₃) ok. MS (FAB)m/e=420 (M+1).

EXAMPLE 9

Z--Leu--Abu--CONH--nBu

This compound was synthesized from the corresponding protectedα-ketoester and butyl amine in 42% yield by the procedure described inExample 1. Single spot on TLC, R_(f) =0.54 (CHCl₃ /CH₃ OH 50:1 ); mp135°-136° C. Anal. calcd. for C₂₃ H₃₅ N₃ O₅ : 433.53; C, 63.71; H, 8.13;N, 9.69. Found: C, 63.48; H, 8.07; N, 9.67. NMR (CDCl₃) ok. MS (FAB)m/e=434 (M+1).

EXAMPLE 10

Z--Leu--Abu--CONH--iBu

This compound was synthesized from the corresponding protectedα-ketoester and isobutylamine in 65% yield by the procedure described inExample 1. Single spot on TLC, R_(f) =0.25 (CHCl₃ /CH₃ OH 50:1); mp133°-135° C. Anal. calcd. for C₂₃ H₃₅ N₃ O₅ : 433.52; C, 63.72; H, 8.14;N, 9.69. Found: C, 63.46; H, 8.10; N, 9.60. NMR (CDCl₃) ok. MS (FAB)m/e=434 (M+1).

EXAMPLE 11

Z--Leu--Abu--CONH--Bzl

This compound was synthesized from the corresponding protectedα-ketoester and benzylamine in 29% yield by the procedure described inExample 5. Single spot on TLC, R_(f) =0.56 (CHCl₃ /CH₃ OH 30:1); mp140°-141° C. Anal. calcd. for C₂₆ H₃₃ N₃ O₅ : 467.54; C, 66.79; H, 7.11;N, 8.99. Found: C, 66.65; H, 7.07; N, 8.93. NMR (CDCl₃) ok. MS (FAB)m/e=468 (M+1).

EXAMPLE 12

Z--Leu--Abu--CONH--(CH₂)₂ Ph

This compound was synthesized from the corresponding protectedα-ketoester and phenethylamine in 51% yield by the procedure describedin Example 5. Single spot on TLC, R_(f) =0.44 (CHCl₃ /CH₃ OH 30:1); mp156°-157° C. Anal. calcd. for C₂₇ H₃₅ N₃ O₅ : 481.59; C, 67.34; H, 7.33;N, 8.72. Found: C, 67.38; H, 7.33; N, 8.78. NMR (CDCl3) ok. MS (FAB)m/e=482 (M+1).

EXAMPLE 13

Z--Leu--Abu--CONH--(CH₂)₃ --N(CH₂ CH₂)₂ O

This compound was synthesized from protected α-ketoester and4(3-aminopropyl)morpholine in 33% yield by the procedure described inExample 1. After reacting overnight, ethyl acetate (80 ml) was added.The mixture was filtered to remove a white precipitate. The solution waswashed with water (3×20 ml), saturated sodium chloride (2×20 ml), anddried over magnesium sulfate. The solution was evaporated leaving ayellow oil. Chromatography on a silica gel column with CHCl₃ /CH₃ OH(10:1 v/v) afforded a yellow semisolid, which was recrystallized fromethyl acetate/hexane to obtain a pale yellow solid. Single spot on TLC,R_(f) =0.42 (CHCl₃ /CH₃ OH 10:1); mp 125°-126° C. Anal. calcd. for C₂₆H₄₀ N₄ O₆ : 504.63; C, 61.88; H, 7.99; N, 11.10. Found: C, 61,69; H,7.95; N, 11.07. NMR (CDCl₃) ok. MS (FAB) m/e=505 (M+1).

EXAMPLE 14

Z--Leu--Abu--CONH--(CH₂)₇ CH₃

This compound was synthesized from the corresponding protectedα-ketoester and octylamine in 67% yield by the procedure described inExample 5. It was while solid. Single spot on TLC, R_(f) =0.55 (CHCl₃/CH₃ OH 30:1); mp 134°-135° C. Anal. calcd. for C₂₇ H₄₃ N₃ O₅ : 489.66;C, 66.23; H, 8.85; N, 8.58. Found: C, 66.19; H, 8.81; N, 8.61. NMR(CDCl₃) ok. MS (FAB) m/e=490 (M+1).

EXAMPLE 15

Z--Leu--Abu--CONH--(CH₂)₂ OH

This compound was synthesized from the corresponding protectedα-ketoester and ethanolamine in 29% yield by the procedure described inExample 13. The product was a white sticky solid. Single spot on TLC,R_(f) =0.42 (CHCl₃ /CH₃ OH 10:1); mp 151°-153° C. Anal: calcd. for C₂₁H₃₁ N₃ O₆ : 421.49; C, 59.84; H, 7.41; N, 9.97. Found: C, 59.11; H,7.44; N, 9.81. NMR (CDCl₃) ok. MS (FAB) m/e=422 (M+1).

EXAMPLE 16

Z--Leu--Abu--CONH--(CH₂)₂ O(CH₂)₂ OH

This compound was synthesized from the corresponding protectedα-ketoester and 2-(2-aminoethoxy)ethanol in 34% yield by the proceduredescribed in Example 13. The product was white sticky solid. Single spoton TLC, R_(f) =0.42 (CHCl₃ /CH₃ OH 10:1); mp 103°-105° C. Anal.: calcd.for C₂₃ H₃₅ N₃ O₇ : 465.55; C, 59.34; H, 7.58; N, 9.03. Found: C, 59.23;H, 7.58; N, 9.01. NMR (CDCl₃) ok. MS (FAB) m/e=466 (M+1).

EXAMPLE 17

Z--Leu--Abu--CONH--(CH₂)₁₇ CH₃

This compound was synthesized from the corresponding protectedα-ketoester and octadecylamine in 12% yield by the procedure describedin Example 5. The product was a pale yellow solid. Single spot on TLC,R_(f) =0.54 (CHCl₃ /CH₃ OH 30:1); mp 134°-136° C. Anal: calcd. for C₃₇H₆₃ N₃ O₅ : 629.92; C, 70.55; H, 10.08; N, 6.67. Found: C, 70.71; H,10.14; N, 6.75. NMR (CDCl₃) ok. MS (FAB) m/e=630.2 (M+1).

EXAMPLE 18

Z--Leu--Abu--CONH--CH₂ --C₆ H₃ (OCH₃)₂

This compound was synthesized from the corresponding protectedα-ketoester and 3,5-dimethoxybenzylamine in 45% yield by the proceduredescribed in Example 5. The product was yellow sticky solid. Single spoton TLC, R_(f) =0.44 (CHCl₃ /CH₃ OH 30:1); mp 153°-155° C. Anal.: calcd.for C₂₈ H₃₇ N₃ O₇ : 527.62; C, 63.74; H, 7.07; N, 7.96. Found: C, 63.66;H, 7.09; N, 7.92. NMR (CDCl₃) ok. MS (FAB) m/e=528.8 (M+1).

EXAMPLE 19

Z--Leu--Abu--CONH--CH₂ --C₄ H₄ N

This compound was synthesized from the corresponding protectedα-ketoester and 4-(aminomethyl)pyridine in 45% yield by the proceduredescribed in Example 13. The product was greenish yellow solid. Singlespot on TLC, R_(f) =0.55 (CHCl₃ /CH₃ OH 10:1); mp 124°-126° C. Anal:calcd. for C₂₅ H₃₂ N₄ O₅ : 468.55; C, 64.08; H, 6.88; N, 11.96. Found:C, 63.88; H, 6.87; N, 11.96. NMR (CDCl₃) ok. MS (FAB) m/e =469 (M+1).

It is obvious that those skilled in the art may make modifications tothe invention without departing from the spirit of the invention or thescope of the subjoined claims and their equivalents.

What is claimed is:
 1. A peptide ketoamide compound of the formula:

    M.sub.1 --AA.sub.2 --AARES--CO--NH--R.sub.4

or a pharmaceutically acceptable salt, wherein AARES represents--NH--CHR₂ --CO--, M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂--, X--NH--CO--, X₂ N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂N--SO₂ --, X--CO--, X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--; X isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ fluoroalkyl,C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkyl substituted with J,1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyldisubstituted with K, phenyl trisubstituted with K, naphthyl, naphthylsubstituted with K, naphthyl disubstituted with K, naphthyltrisubstituted with K, C₁₋₁₀ alkyl with an attached phenyl group, C₁₋₁₀alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with an attachedphenyl group substituted with K, C₁₋₁₀ alkyl with two attached phenylgroups substituted with K, C₁₋₁₀ alkyl with an attached phenoxy group,and C₁₋₁₀ alkyl with an attached phenoxy group substituted with K on thephenoxy group; J is selected from the group consisting of halogen, COOH,OH, CN, NO₂, NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine,C₁₋₁₀ alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--; K isselected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfloroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁₋₁₀ acyl, C₁₋₁₀ alkoxy-CO--, and C₁₋₁₀alkyl-S--; AA₂ is a side chain blocked or unblocked amino acid with theL configuration, D configuration, or no chirality at the α-carbonselected from the group consisting of alanine, valine, leucine,isoleucine, proline, methionine, methionine sulfoxide, phenylalanine,tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine; R₂ is selected from the groupconsisting of C₁₋₈ branched and unbranched alkyl, C₁₋₈ branched andunbranched cyclized alkyl, and C₁₋₈ branched and unbranched fluoroalkyl;R₄ is selected from the group consisting of C₃₋₂₀ cyclized alkyl with anattached phenyl group, C₁₋₂₀ alkyl with an attached phenyl groupsubstituted with K, C₁₋₂₀ alkyl with an attached phenyl groupdisubstituted with K, C₁₋₂₀ alkyl with an attached phenyl grouptrisubstituted with K, C₃₋₂₀ cyclized alkyl with an attached phenylgroup substituted with K, C₁₋₁₀ alkyl with a morpholine ring attachedthrough nitrogen to the alkyl, C₁₋₁₀ alkyl with a piperidine ringattached through nitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidinering attached through nitrogen to the alkyl, C₁₋₂₀ alkyl with an OHgroup attached to the alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ alkyl with anattached 4-pyridyl group, C₁₋₁₀ alkyl with an attached 3-pyridyl group,C₁₋₁₀ alkyl with an attached 2-pyridyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).
 2. A peptideketoamide compound of the formula:

    M.sub.1 --AA2--AARES--CO--NH--R.sub.

or a pharmaceutically acceptable salt, wherein AARES represents--NH--CHR₂ --CO--, M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂--, X--NH--CO--, X₂ N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂N--SO₂ --, X--CO--, X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--; X isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ fluoroalkyl,C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkyl substituted with J,1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyldisubstituted with K, phenyl trisubstituted with K, naphthyl, naphthylsubstituted with K, naphthyl disubstituted with K, naphthyltrisubstituted with K, C₁₋₁₀ alkyl with an attached phenyl group, C₁₋₁₀alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with an attachedphenyl group substituted with K, C₁₋₁₀ alkyl with two attached phenylgroups substituted with K, C₁₋₁₀ alkyl with an attached phenoxy group,and C₁₋₁₀ alkyl with an attached phenoxy group substituted with K on thephenoxy group; J is selected from the group consisting of halogen, COOH,OH, CN, NO₂, NH₂, C₁₋₁₀ alkoxy, C₂₋₁₂ to alkylamine, C₂₋₁₂ dialkylamine,C₁₋₁₀ alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--; K isselected from the group consisting of halogen, C₁₋₁₀ perfluoroalkyl,C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀ to alkylamino, C₂₋₁₂dialkylamino, C₁ -C₁₀ acyl, C₁₋₁₀ alkoxy-CO--, and C₁₋₁₀ alkyl-S--; AA₂is a side chain blocked or unblocked amino acid with the Lconfiguration, D configuration, or no chirality at the α-carbon selectedfrom the group consisting of alanine, valine, leucine, isoleucine,proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt2)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine, and hexafluoroleucine; R₂ is selected from the groupconsisting of C₁₋₈ branched and unbranched alkyl, C₁₋₈ branched andunbranched cyclized alkyl, and C₁₋₈ branched and unbranched fluoroalkyl;R₄ is selected from the group consisting of C₃₋₂₀ cyclized alkyl with anattached phenyl group, C₁₋₂₀ alkyl with an attached phenyl groupsubstituted with K, C₃₋₂₀ alkyl with an attached phenyl groupdisubstituted with K, C₁₋₂₀ alkyl with an attached phenyl grouptrisubstituted with K, C₃₋₂₀ cyclized alkyl with an attached phenylgroup substituted with K, C₁₋₁₀ alkyl with a morpholine ring attachedthrough nitrogen to the alkyl, C₁₋₁₀ alkyl with a piperidine ringattached through nitrogen to the alkyl, C₁₋₁₀ alkyl with a pyrrolidinering attached through nitrogen to the alkyl, C₁₋₂₀ alkyl with an OHgroup attached to the alkyl, --CH₂ CH₂ OCH₂ CH₂ OH, C₁₋₁₀ alkyl with anattached 4-pyridyl group, C₁₋₁₀ alkyl with an attached 3-pyridyl group,C₁₋₁₀ alkyl with an attached 2-pyridyl group, --NH--CH₂ CH₂--(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).
 3. A peptideketoamide compound of the formula:

    M.sub.1 --AA.sub.2 --AARES--CO--NH--R.sub.4

or a pharmaceutically acceptable salt, wherein AARES represents--NH--CHR₂ --CO--, M₁ represents H, NH₂ --CO--, NH₂ --CS--, NH₂ --SO₂--, X--NH--CO--, X₂ N--CO--, X--NH--CS--, X₂ N--CS--, X--NH--SO₂ --, X₂N--SO₂ --, X--CO--, X--CS--, X--SO₂ --, X--O--CO--, or X--O--CS--; X isselected from the group consisting of C₁₋₁₀ alkyl, C₁₋₁₀ fluoroalkyl,C₁₋₁₀ alkyl substituted with J, C₁₋₁₀ fluoroalkyl substituted with J,1-admantyl, 9-fluorenyl, phenyl, phenyl substituted with K, phenyldisubstituted with K, phenyl trisubstituted with K, naphthyl, naphthylsubstituted with K, naphthyl disubstituted with K, naphthyltrisubstituted with K, C₁₋₁₀ alkyl with an attached phenyl group, C₁₋₁₀alkyl with two attached phenyl groups, C₁₋₁₀ alkyl with an attachedphenyl group substituted with K, C₁₋₁₀ alkyl with two attached phenylgroups substituted with K, C₁₋₁₀ alkyl with an attached phenoxy group,and C₁₋₁₀ alkyl with an attached phenoxy group substituted with K on thephenoxy group; J is selected from the group consisting of halogen, COOH,OH, CN, NO₂, NH₂, C₁₋₁₀ alkoxy, C₁₋₁₀ alkylamine, C₂₋₁₂ dialkylamine,C₁₋₁₀ alkyl-O--CO--, C₁₋₁₀ alkyl-O--CO--NH--, and C₁₋₁₀ alkyl-S--; K isselected from the group consisting of halogen, C₁₋₁₀ alkyl, C₁₋₁₀perfluoroalkyl, C₁₋₁₀ alkoxy, NO₂, CN, OH, CO₂ H, amino, C₁₋₁₀alkylamino, C₂₋₁₂ dialkylamino, C₁ -C₁₀ acyl, C₁₋₁₀ -alkoxy-CO--, andC₁₋₁₀ alkyl-S--; AA₂ is a side chain blocked or unblocked amino acidwith the L configuration, D configuration, or no chirality at theα-carbon selected from the group consisting of alanine, valine, leucine,isoleucine, proline, methionine, methionine sulfoxide, phenylalanine,tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine,glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine,phenylglycine, beta-alanine, norleucine, norvaline, alpha-aminobutyricacid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine,homoarginine, sarcosine, indoline 2-carboxylic acid,2-azetidinecarboxylic acid, pipecolinic acid (2-piperidine carboxylicacid), O-methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine,S-benzylcysteine, NH₂ --CH(CH₂ CHEt₂)-COOH, alpha-aminoheptanoic acid,NH₂ --CH(CH₂ -1-napthyl)-COOH, NH₂ --CH(CH₂ -2-napthyl)-COOH, NH₂--CH(CH₂ -cyclohexyl)-COOH, NH₂ --CH(CH₂ -cyclopentyl)-COOH, NH₂--CH(CH₂ -cyclobutyl)-COOH, NH₂ --CH(CH₂ -cyclopropyl)-COOH,trifluoroleucine and hexafluoroleucine; R₂ is selected from the groupconsisting of C₁₋₈ branched and unbranched alkyl, C₁₋₈ branched andunbranched cyclized alkyl, and C₁₋₈ branched and unbranched fluoroalkyl;R₄ is selected from the group consisting of C₁₋₁₀ alkyl with amorpholine ring attached through nitrogen to the alkyl, C₁₋₁₀ alkyl witha piperidine ring attached through nitrogen to the alkyl, C₁₋₁₀ alkylwith a pyrrolidine ring attached through nitrogen to the alkyl, C₁₋₂₀alkyl with an OH group attached to the alkyl, --CH₂ CH₂ OCH₂ CH₂ OH,C₁₋₁₀ alkyl with an attached 4-pyridyl group, C₁₋₁₀ alkyl with anattached 3-pyridyl group, C₁₋₁₀ alkyl with an attached 2-pyridyl group,--NH--CH₂ CH₂ --(4-hydroxyphenyl), and --NH--CH₂ CH₂ --(3-indolyl).